Source of the article: Molecular Diagnostic Laboratory
Author: Research and Development Xiaozhi
Primer design for PCR (polymerase chain reaction) is a crucial step in molecular biology experiments, and its quality directly affects the specificity and efficiency of PCR reactions. The following are 12 golden rules for PCR primer design. These rules are based on extensive experimental experience and scientific principles, aiming to assist researchers in designing efficient and specific PCR primers.
1. Conservative zone designPrinciplePrimers are best designed within the conserved region of the template cDNA. The conserved regions of DNA sequences are determined by comparing similar sequences among species.ExplanationThe conserved region is a relatively stable area in the gene sequence that is less prone to variation. Designing primers in the conserved region can ensure the specific binding of primers to the target sequence and reduce the risk of non-specific amplification.PrinciplePrimer lengths are generally between 15 and 30 bases, with 18 to 27 bp being the most commonly used, but should not exceed 38 bp.ExplanationIf the primer length is too short, it may reduce the specific binding ability to the template DNA, while if it is too long, it will cause the extension temperature to be too high, which is not conducive to the reaction of Taq DNA polymerase. Therefore, choosing the appropriate primer length is crucial for the success of PCR reactions.PrincipleThe GC content of primers should be between 40% and 60%, and the Tm value is preferably close to 72℃.ExplanationBoth excessively high and low GC content will affect the initiation efficiency of primers. The GC content of the upstream and downstream primers should be kept consistent to ensure that they have similar unchain temperatures (Tm values). The Tm value is the temperature at which the primer and the template DNA are half unchained, reflecting the stability of the binding between the primer and the template DNA. An appropriate Tm value helps to enhance the specificity and efficiency of PCR reactions.4. Avoid the third bit of the codonPrincipleThe 3' end of the primer should avoid the third bit of the codon.ExplanationIf the encoding region is to be amplified, the 3' end of the primer should not terminate at the 3rd position of the codon, as the 3rd position of the codon is prone to degeneracy (i.e., multiple codons encoding the same amino acid), which will affect the specificity and efficiency of amplification.PrincipleThe 3' end of the primer cannot be selected as A; it is best to choose T.ExplanationWhen the 3' end of the primer is mismatched, there is a significant difference in the initiation efficiency among different bases. When the last base is A, chain synthesis can be initiated even in the case of mismatch. However, when the last digit is T, the triggering efficiency of mismatch is greatly reduced. Therefore, in order to enhance the specificity of the PCR reaction, it is best to choose T at the 3' end of the primer.6. Bases are randomly distributedPrincipleThe distribution of bases in primers should be as random as possible to avoid the presence of polypurines or polypyrimidines.ExplanationThe presence of polypurines or polypyrimidines may cause primers to form incorrect binding sites on the template DNA, thereby reducing the specificity of the PCR reaction. Therefore, when designing primers, it is necessary to ensure that the bases are randomly distributed and avoid consecutive G or C.7. Avoid complementary sequencesPrincipleThere should be no complementary sequences between primers themselves or between primers.ExplanationThe complementary sequences of primers themselves will form Hairpin structures, and this secondary structure will affect the retaliatory binding of primers to template DNA due to steric hindrance. The two primers should not be complementary either, especially the complementary overlap at the 3' end should be avoided to prevent the formation of primer dimers (Dimer and Cross dimer). All these will reduce the specificity and efficiency of the PCR reaction.PrincipleThe ΔG values at the 5' end and the middle of the primer should be relatively high, while the ΔG value at the 3' end should be relatively low.ExplanationThe ΔG value refers to the free energy required for the formation of a double-stranded DNA, which reflects the relative stability of the base pairs within the double-stranded structure. Selecting primers with relatively high ΔG values at the 5' end and the middle, while having a lower ΔG value at the 3' end, helps ensure the specific binding of primers to template DNA and prevent the occurrence of non-specific amplification.The 9.5 'end can be modifiedPrincipleThe 5' end of the primer can be modified, but the 3' end cannot.ExplanationThe 5' end of the primer determines the length of the PCR product and has little effect on amplification specificity. Therefore, modifications can be made at the 5' end to introduce enzyme digestion sites, fluorescent labeling, etc. for subsequent experimental operations. The extension of primers starts from the 3' end, so the 3' end cannot be modified in any way and there is no possibility of forming a secondary structure.10. Avoid the secondary structure areaPrincipleThe single strand of the amplification product cannot form a secondary structure.ExplanationThe main reason for the ineffectiveness of some primers is the influence of the single-stranded secondary structure of the amplification products. When choosing amplification fragments, it is best to avoid secondary structure regions. The stable secondary structure of mRNA can be predicted using relevant software (such as RNAstructure), which is helpful for selecting the appropriate template region for amplification.Principle：After the primer design is completed, BLAST testing should be conducted to ensure its specificity.ExplanationBLAST detection can assess the similarity of primers to other gene sequences by comparing them with sequences in a known gene sequence database. If the primers have a high degree of similarity to other gene sequences, it may lead to non-specific amplification. Therefore, specific detection is an important step to ensure the effectiveness of primers.12. Take the experimental conditions into comprehensive considerationPrincipleWhen designing PCR primers, it is also necessary to comprehensively consider experimental conditions such as annealing temperature and Mg2 +Concentration, etc.ExplanationDifferent experimental conditions may affect the specificity and efficiency of PCR reactions. Therefore, when designing primers, appropriate adjustments should also be made according to the experimental conditions to ensure the successful progress of the PCR reaction. For instance, parameters such as the length of primers, GC content and Tm value can be adjusted according to the experimental conditions to optimize the PCR reaction conditions.
